Other entities represented in this entry:
HGNC Approved Gene Symbol: DUX4
Cytogenetic location: 4q35.2 Genomic coordinates (GRCh38) : 4:190,173,774-190,185,911 (from NCBI)
DUX4 Gene
DUX4 encodes a putative transcription factor that contains 2 homeobox domains. A copy of DUX4 is found within each of up to 100 copies of the D4Z4 macrosatellite repeat in the subtelomeric region of chromosome 4q. Only the last repeat appears to encode a functional DUX4 transcript with a polyadenylation site that lies telomeric and outside the D4Z4 repeat region (summary by Snider et al., 2010).
D4Z4 Microsatellite Repeat
Multiple copies of a 3.3-kb repeat, designated D4Z4, exist in the proximal subtelomeric region of 4q35. Each repeat contains a copy of DUX4. The polymorphic D4Z4 repeat array is composed of 11 to 150 repeats in normal individuals. Most patients with facioscapulohumeral muscular dystrophy (FSHD; 158900) carry a D4Z4 repeat array of less than 11 repeats. A similar subtelomeric repeat array configuration occurs on chromosome 10qter (summary by van Overveld et al., 2005).
While sequencing the 3.3-kb repeat elements remaining in an FSHD patient, Gabriels et al. (1999) identified a putative promoter for an ORF encompassing the 2 homeoboxes reported by Hewitt et al. (1994) and named the putative gene DUX4. DUX4 encodes a deduced 391-amino acid protein containing 2 homeodomains. Gabriels et al. (1999) found an identical copy of DUX4 in each of the 3.3-kb repeat elements remaining in the FSHD patient. Using in vitro transcription/translation, they detected products with apparent molecular masses of 38 kD and 75 kD on SDS-PAGE, corresponding to the DUX4 monomer and dimer, respectively.
By PCR of FSHD primary myoblast RNA, Dixit et al. (2007) identified 2 DUX4 splice variants that differed due to inclusion or exclusion of an intron in their 3-prime UTRs. The deduced DUX4 protein contains 424 amino acids, larger than the protein initially reported (Gabriels et al., 1999) due to corrections of sequencing errors. PCR analysis detected the 5-prime end of DUX4 mRNA in both FSHD and control myoblasts, whereas the 3-prime end specific to the most distal unit of DUX4, which includes a polyadenylation sequence, was detected only in FSHD myoblasts. Western blot analysis of transfected cells revealed a 52-kD DUX4 protein. DUX4 protein was detected in FSHD samples, but not controls. Dixit et al. (2007) concluded that only the DUX4 gene in the most distal D4Z4 element is likely to be transcribed into a polyadenylated RNA and to be translated in vivo.
Kowaljow et al. (2007) found that DUX4 transcripts had only short poly(A) tails and did not fractionate with mRNAs containing long poly(A) tails. RT-PCR detected DUX4 expression in cultured FSHD patient myoblasts and in human fetal and adult rhabdomyosarcoma cell lines, but not in control myoblasts. Immunofluorescence analysis of DUX4-transfected cell lines showed a nuclear staining pattern that partly overlapped with the nuclear envelope.
Snider et al. (2009) found that the D4Z4 region is subject to highly complex splicing and mRNA processing. Sequencing of the 2 residual D4Z4 repeats in an individual with FSHD and the disease-associated haplotype 4qA161 suggested the presence of major and minor ORFs, including the DUX4 ORF, that overlap in different reading frames and in both orientations. Many of the potential ORFs in the 2 D4Z4 repeats, but not the DUX4 ORF, differ significantly due to sequence variations. RT-PCR confirmed expression of full-length DUX4 transcripts at low levels in human embryonic and mesenchymal stem cells, and DUX4 protein was detected in undifferentiated human embryonic and mesenchymal stem cells. However, transcripts including only the 5-prime or 3-prime portions of DUX4 were detected at higher abundance than the full-length transcript in fibroblasts and muscle cells from FSHD and control individuals. Transcripts containing only the 5-prime portion of the DUX4 ORF were polyadenylated and capped in FSHD muscle cells, and they were predicted to encode a truncated protein containing only the paired homeodomains. The majority of these transcripts had a repeated sequence, suggesting splicing between the 2 D4Z4 units. Transcripts containing only the 3-prime portion of the DUX4 ORF were polyadenylated and uncapped in FSHD muscle cells, and they were predicted to encode a 76-amino acid protein containing only the highly conserved C-terminal region. Transfection studies demonstrated expression of the DUX4 isoform containing only the C-terminal domain and suggested that it originated from an internal ribosomal initiation site. RT-PCR also detected several sense and antisense transcripts originating from the D4Z4 repeats in muscle cells from FSHD and control individuals. Northern blot analysis revealed that microRNA-like fragments were generated from the DUX4 transcript in FSHD and control fibroblasts and muscle cells.
Snider et al. (2010) stated that they had previously identified a short DUX4 transcript (DUX4-s), generated by splicing at a noncanonical donor sequence. The deduced protein contains 2 homeobox domains, but lacks the C-terminal end found in full-length DUX4 (DUX4-fl). RT-PCR of normal human tissues detected abundant DUX4-fl expression in testis, but not in any other tissue examined. DUX4-s was detected in ovary, heart, and skeletal muscle, with weaker expression in liver. No DUX4-s expression was detected in testis, kidney, or cerebellum. Within cells and cell lines, DUX4-fl was detected in induced pluripotent stem cells and in some human embryonic stem cell lines. Differentiated fibroblasts and embryoid bodies expressed DUX4-s only. Immunohistochemical analysis of human testis detected DUX4-fl in spermatogonia and primary spermatocytes and in more differentiated cells in seminiferous tubules.
Dixit et al. (2007) reported that the DUX4 promoter region contains putative CATT, GC, TACAA, and E boxes. The 3-prime end of the DUX4 gene contains an alternatively spliced exon located between the stop codon and the end of the D4Z4 unit. In addition, the 3-prime end of the DUX4 gene in the most distal D4Z4 unit contains a second intron and a single polyadenylation signal, both of which are located telomeric to the D4Z4 unit.
Snider et al. (2010) determined that the DUX4 gene contains at least 7 exons, with exons 3 through 7 outside the D4Z4 repeat region. The 4qA haplotype includes alternate polyadenylation sites in exons 3 and 7. The 4qB haplotype does not show these polyadenylation sites and may include more than 7 exons.
By genomic sequence analysis, Gabriels et al. (1999) mapped the DUX4 gene to chromosome 4q35.
Using luciferase reporter assays, Gabriels et al. (1999) demonstrated DUX4 promoter activity that was dependent on a TACAA box and a GC box. They suggested that DUX4 may play a role in FSHD because partial deletions of the D4Z4 region might alter DUX4 expression in FSHD patients.
By comparing genomewide expression profiles of muscle biopsies from patients with FSHD to those of 11 other neuromuscular disorders, Dixit et al. (2007) identified 5 genes, including the paired homeodomain transcription factor PITX1 (602149), that were specifically upregulated in FSHD patients. DUX4 expression was upregulated in FSHD myoblasts at both the mRNA and protein levels. DUX4 activated expression of a reporter gene fused to the PITX1 promoter and of endogenous Pitx1 in DUX4-transfected C2C12 mouse myoblasts. In electrophoretic mobility shift assays, DUX4 specifically interacted with a 30-bp sequence containing a conserved TAAT core motif in the PITX1 promoter. Mutations of the TAAT core affected Pitx1 activation in C2C12 cells and DUX4 binding in vitro.
Kowaljow et al. (2007) found that overexpression of DUX4 in several cell lines resulted in apoptotic changes, including activation of caspase-3 (CASP3; 600636) and caspase-7 (CASP7; 601761).
Using transfection studies, Snider et al. (2009) showed that full-length DUX4 reduced the expression of reporter genes driven by CKM (123310), desmin (DES; 125660), or a multimerized E box. Full-length DUX4 also reduced expression of endogenous myosin heavy chain (see 160730) in transfected C2C12 mouse myoblasts. Expression of the full-length DUX4 protein or isoforms containing only the N- or C-terminal domains inhibited myogenesis in C2C12 cells. Several DUX4 transcripts incapable of translation also inhibited C2C12 differentiation. Expression of transcripts encoding full-length DUX4 inhibited zebrafish development past gastrulation or caused severe developmental abnormalities in the surviving embryos. Expression of transcripts encoding the C-terminal DUX4 peptide did not alter morphologic development of zebrafish, but blocked myogenesis at a step between Myod (159970) transcription and activation of Myod target genes.
Wallace et al. (2011) showed that apoptotic changes observed in DUX4-expressing HEK293 cells were eliminated by an inactivating mutation in the first DNA-binding homeobox domain of DUX4. The development of lesions in DUX4-injected mouse muscle was also abrogated by mutation of the DUX4 homeobox domain. Pharmacologic inhibition of p53 (TP53; 191170) mitigated DUX4 toxicity in HEK293 cells, and muscle from p53-null mice were resistant to DUX4-induced damage. Wallace et al. (2011) concluded that DUX4-induced myopathy is dependent on p53-mediated apoptosis.
By gene expression array analysis, Geng et al. (2012) found that DUX4-fl induced expression of several genes associated with germline and early stem-cell development and suppressed genes involved in innate immune response. DUX4-fl bound specifically to a long terminal repeat element from a class of MaLR retrotransposons. DUX4-s bound the same element, but it did not activate gene expression, and it functioned as a dominant-negative inhibitor of DUX4-fl when coexpressed. Although DUX4 expression was below the level of detection in several FSHD muscle biopsy samples by RT-PCR, expression of DUX4-fl-regulated genes was significantly upregulated compared with control muscle samples. Furthermore, knockdown of DUX4-fl expression in FSHD muscle cells via small interfering RNA or coexpression of DUX4-s reduced the expression of DUX4-fl target genes. DEFB103A (606611) and DEFB103B, which encode negative regulators of innate immunity, were among the DUX4-fl target genes upregulated in FSHD muscle. Overexpression of DEFB103 in differentiating muscle cells reduced expression of genes associated with muscle differentiation and inflammatory response. Geng et al. (2012) concluded that even low-level DUX4 overexpression can have a profound effect on gene expression and result in FSHD, and they proposed that DEFB103 may contribute to FSHD pathology.
D4Z4 Macrosatellite Repeat
Hewitt et al. (1994) determined the sequence of D4Z4 and showed that each copy of the repeat contains 2 homeoboxes and 2 previously described repetitive sequences, LSau and a GC-rich low copy repeat designated hhspm3. By Southern blot analysis, FISH, and isolation of cDNA and genomic clones, Hewitt et al. (1994) showed that there are repeat sequences similar to D4Z4 at other locations in the human genome. Southern blot analysis of primate genomic DNA indicated that the copy number of D4Z4-like repeats has increased markedly within the last 25 million years. Two cDNA clones were isolated and found to contain stop codons and frameshifts within the homeodomains. An STS was produced to the cDNAs, and analysis of a somatic cell hybrid panel suggested that they mapped to chromosome 14. No cDNA clones mapping to the 4q35 D4Z4 repeat were identified, although the possibility that these repeats encode a protein could not be ruled out. Although D4Z4 may not encode a protein, there is association between deletions within this locus and FSHD1 (158900). The D4Z4 repeats contain LSau repeats and are adjacent to 68-bp Sau3A repeats. Both of these sequences are associated with heterochromatic regions of DNA, regions known to be involved in the phenomenon of 'position effect' variegation. Hewitt et al. (1994) postulated that deletion of D4Z4 sequences produced a position effect that is involved in the pathogenesis of FSHD.
Winokur et al. (1993) also postulated that FSHD may be due to a position effect. Bengtsson et al. (1994) reported results indicating that the tandem array of 3.2-kb repeats, disrupted in FSHD, lies immediately adjacent to the telomere of 4q and that the gene responsible for FSHD is probably located proximal to the tandem repeat.
A highly homologous polymorphic repeat array is located near the telomere of 10q, but a specific BlnI site within each chromosome 10-derived repeat unit allows discrimination between the arrays (Deidda et al., 1996). This BlnI site-dependent discrimination demonstrated the presence of 10-type repeats on chromosome 4 and, vice versa, 4-type repeats on chromosome 10, suggesting a dynamic exchange between these chromosomes. Van der Maarel et al. (2000) found that the repeat deletion was significantly enhanced by supernumerary homologous repeat arrays. They demonstrated that a numerical excess of 4-type repeats on chromosome 10 was a significant, if not the major, predisposing factor for the occurrence of the FSHD-type deletion. Mitotic interchromosomal gene conversion or translocation between fully homologous repeat arrays may be a major mechanism for FSHD mutations.
Van Deutekom et al. (1996) reported exchange of subtelomeric repeated DNA units between chromosomes 4q35 and 10q26 in at least 20% of the Dutch population. These subtelomeric rearrangements generated novel DNA restriction fragments and complicated the use of restriction fragment analysis for DNA diagnosis of FSHD. The high frequency of interchromosomal exchanges of 3.3-kb repeat units suggested to van Deutekom et al. (1996) that these units probably do not contain part of the FSHD gene and supported position effect variegation as the most likely mechanism for FSHD.
Using 3-dimensional immuno-FISH, Masny et al. (2004) determined that the FSHD region at chromosome 4q35.2 localized to the nuclear periphery in several cell types throughout the cell cycle. FSHD region chromatin localization to the nuclear envelope was lost in lamin A/C (150330)-null fibroblasts, suggesting that lamin A/C is required for proper localization, and both normal and D4Z4-deleted alleles localized to the nuclear periphery. Masny et al. (2004) suggested that FSHD likely arises from improper interactions with transcription factors or chromatin modifiers at the nuclear envelope.
Within the D4Z4 locus, Petrov et al. (2006) identified 2 DNA loop domains anchored to the nuclear matrix via nuclear scaffold/matrix attached regions (S/MARs). Myoblasts derived from patients with FSHD showed a significant decrease in association of S/MARs with the nuclear matrix compared to control myoblasts. Biochemical mapping showed that in normal myoblasts the D4Z4 array was located in a DNA loop domain distinct from the DNA loop domain where FRG1 (601278) and FRG2 (609032) were located, whereas in damaged FSHD chromosome, the partially deleted D4Z4 array and FRG1 and FRG2 were located within the same DNA loop domain. Petrov et al. (2006) suggested that S/MARs regulate chromatin accessibility and expression of genes implicated in FSHD.
Petrov et al. (2008) proposed that FR-MAR, an S/MAR positioned 5-prime of the D4Z4 repeat array, may function in normal cells as an insulator element to protect upstream genes from the effect of D4Z4. Using reporter gene assays, they found that D4Z4 repeats showed enhancer function and elevated transcription from the FRG1 promoter in all transfected cell lines examined. Deletion analysis located the strongest enhancer activity to the 5-prime end of the D4Z4 unit. FR-MAR blocked this enhancer function. FR-MAR also associated with the nuclear matrix in normal myoblasts, but not in FSHD myoblasts. Petrov et al. (2008) concluded that FR-MAR functions as an insulator to protect the FRG genes from the enhancer activity present in each D4Z4 repeat unit.
By in vitro cellular studies with a single D4Z4 repeat, Ottaviani et al. (2009) demonstrated that D4Z4 acts both as a transcriptional insulator protecting against the repressive influence of various chromosomal contexts and as an enhancer insulator interfering with enhancer-promoter communication. The addition of D4Z4 element repeats progressively abolished the insulation activity, suggesting that the repeat element loosens its anti-silencing activity upon multimerization. Further studies showed that the insulator function of D4Z4 is dependent on CTCF (604167) and LMNA (150330). The findings demonstrated a novel mode of chromatin regulation controlled by the number of D4Z4 repeats. Ottaviani et al. (2009) proposed that reduction of the D4Z4 array in FSHD patients results in a gain-of-function effect by allowing the binding of CTCF and provoking changes in the biologic function of D4Z4 such that it switches from a repressor to an insulator protecting the expression of the FSHD gene(s).
Dmitriev et al. (2011) showed that KLF15 (606465) bound an enhancer element within the D4Z4 repeat unit. Binding of KLF15 to 2 sites within the D4Z4 enhancer drove expression of FRG2 and DUX4C (DUX4L9; 615581), which are located over 40 kb centromeric to the D4Z4 repeat array. KLF15 expression was upregulated following differentiation of normal human myoblasts and following expression of MYOD (159970), and it was upregulated in FSHD myoblasts, myotubes, and muscle biopsies. FSHD cells also showed upregulated expression of MYOD and the KLF15 target gene PPARG (601487), in addition to DUX4C and FRG2. Dmitriev et al. (2011) concluded that MYOD-dependent KLF15 expression is involved in partial activation of the differentiation program in FSHD myoblasts.
4qA and 4qB Polymorphic Segment
Human 4qter and 10qter share a high degree of similarity, including the D4Z4 repeat array; however, contractions affecting the 10qter repeat are nonpathogenic. Van Geel et al. (2002) detected a polymorphic segment of 10 kb directly distal to D4Z4, which they called alleles 4qA and 4qB. Lemmers et al. (2002) reported that although the 2 alleles are equally common in the general population, FSHD is associated solely with the 4qA allele. They suggested that this was the first example of an intrinsically benign subtelomeric polymorphism predisposing to the development of human disease.
Lemmers et al. (2004) concluded that contractions of D4Z4 on 4qB subtelomeres do not cause FSHD. The 2 allelic variants of 4q, 4qA and 4qB, exist in the region distal to D4Z4. Although both variants are almost equally present in the population, FSHD is associated exclusively with the 4qA allele. Lemmers et al. (2004) identified 3 families with FSHD in which each proband carried 2 FSHD-sized alleles and was heterozygous for the 4qA/4qB polymorphism. Segregation analysis demonstrated that FSHD-sized 4qB alleles are not associated with disease, since these were present in unaffected family members. Thus, in addition to a contraction of D4Z4, additional cis-acting elements on 4qA may be required for the development of FSHD. Alternatively, 4qB subtelomeres may contain elements that prevent FSHD pathogenesis.
Lemmers et al. (2007) hypothesized that allele-specific sequence differences among 4qA, 4qB, and 10q alleles underlie the 4qA specificity of FSHD. By examining sequence variations in the FSHD locus, they demonstrated that the subtelomeric domain of 4q can be subdivided into 9 distinct haplotypes, of which 3 carry the distal 4qA variation. They showed that repeat contractions in 2 of the 9 haplotypes, 1 of which is a 4qA haplotype, are not associated with FSHD. They showed that each of these haplotypes has its unique sequence signature, and proposed that specific SNPs in the disease haplotype are essential for the development of FSHD.
Changes in Gene Expression Related to D4Z4
Van Deutekom et al. (1996) identified the FRG1 gene that mapped 100 kb centromeric of the repeated units on chromosome 4q35 that are deleted in FSHD. They identified a polymorphism in exon 1 of this gene and used RT-PCR to amplify reverse transcribed mRNA from lymphocytes and muscle biopsies of patients and controls. These studies indicated that both alleles were transcribed and gave no evidence of 'position effect' variegation leading to repression of allelic transcription.
Gabellini et al. (2002) found that in FSHD muscle, genes located upstream of D4Z4 on 4q35, including FRG1, FRG2, and ANT1 (103220), are inappropriately overexpressed. They showed that an element within D4Z4 specifically binds a multiprotein complex consisting of transcriptional repressor YY1 (600013), HMGB2 (163906), and nucleolin (NCL; 164035). This multiprotein complex binds D4Z4 in vitro and in vivo and mediates transcriptional repression of 4q35 genes. Gabellini et al. (2002) proposed that deletion of D4Z4 leads to the inappropriate transcriptional derepression of 4q35 genes, resulting in disease. In normal individuals, the presence of a threshold number of D4Z4 repeats leads to repression of 4q35 genes by virtue of the DNA-bound multiprotein complex that actively suppresses gene expression. In FSHD patients, deletion of an integral number of D4Z4 repeats reduces the number of bound repressor complexes and consequently decreases or abolishes transcriptional repression of 4q35 genes.
Jiang et al. (2003) found that H4 acetylation levels of a nonrepeated region adjacent to the 4q35 and 10q26 D4Z4 arrays in normal and FSHD lymphoid cells were like those in unexpressed euchromatin, rather than like constitutive heterochromatin. The control and FSHD cells also displayed similar H4 hyperacetylation (like that of expressed genes) at the 5-prime regions of 4q35 candidate genes FRG1 and ANT1. There was no position-dependent increase in transcript levels from these genes in FSHD skeletal muscle samples compared with controls. Jiang et al. (2003) proposed a model for FSHD in which differential long-distance cis looping depends upon the presence of a 4q35 D4Z4 array with less than a threshold number of copies of the 3.3-kb repeat.
Perini and Tupler (2006) suggested that FSHD might be considered a useful model for the study of position effect in humans. The D4Z4 deletion might result in stochastic variation in gene expression in muscle cells and explain the asymmetric involvement of muscles, the great variability of clinical expression between and within families, and the apparent threshold effect whereby there is a requirement for the deletion of a certain number of copies of D4Z4 to develop FSHD.
Osborne et al. (2007) detected no change in expression of the FRG1, FRG2, or ANT1 genes in muscle biopsies from 19 FSHD patients compared to controls. Further studies of the 8-Mb region proximal to the D4Z4 array showed no significant changes in gene expression, no evidence of a position effect, and no evidence of unequal allele-specific expression. However, microarray analysis of global gene expression in FSHD muscle identified 11 upregulated genes with a role in vascular smooth muscle or endothelial cells, suggesting a possible link between muscular dystrophy and vasculopathy in FSHD.
Bosnakovski et al. (2008) conditionally expressed cDNAs for FSHD candidate genes within the D4Z4 repeat, DUX4, FRG1, FRG2, and ANT1, in mouse C2C12 myoblasts at both high and low expression levels and found that only DUX4 was overtly toxic, as indicated by cellular ATP content, morphologic changes, and apoptosis. DUX4 showed variable toxicity when expressed in mouse fibroblasts or embryoid bodies. DUX4 localized to C2C12 cell nuclei within 2 hours of induction. Microarray analysis revealed altered expression in a broad range of genes, with greatest changes in those involved in growth and development and signal transduction. Expression of Myod was also downregulated at an early time point. Oxidative stress and heat shock genes were downregulated at later time points, suggesting that they may be secondary targets. The DUX4 homeodomains are most similar to those of PAX3 (606597) and PAX7 (167410), and overexpression of these genes rescued viability and proliferation in DUX4-expressing C2C12 cells. Bosnakovski et al. (2008) concluded that DUX4 may cause FSHD by interfering with normal PAX3 or PAX7 function in muscle satellite cells.
Lemmers et al. (2010) showed that FSHD patients carry specific single-nucleotide polymorphisms in the chromosomal region distal to the last D4Z4 repeat. This FSHD-predisposing configuration creates a canonic polyadenylation signal for transcripts derived from DUX4, a double homeobox gene that straddles the last repeat unit and the adjacent sequence. Transfection studies revealed that DUX4 transcripts are efficiently polyadenylated and are more stable when expressed from permissive chromosomes. Lemmers et al. (2010) concluded that their findings suggest that FSHD arises through a toxic gain of function attributable to the stabilized distal DUX4 transcript.
Cabianca et al. (2012) found that derepression of chromosome 4q35 genes in FSHD cells was associated with expression of a long noncoding RNA, DBET (614865), from the FSHD locus. DBET recruited the histone methyltransferase Ash1l (ASH1; 607999) for gene repression in an apparent positive-feedback loop.
D4Z4 and Facioscapulohumeral Muscular Dystrophy 2
In affected members of 15 (79%) of 19 families with facioscapulohumeral muscular dystrophy-2 (FSHD2; 158901), Lemmers et al. (2012) identified heterozygous loss-of-function mutations in the SMCHD1 gene (see, e.g., 614982.0001-614982.0005). The mutations in 7 families were initially identified by exome sequencing and confirmed by Sanger sequencing. The mutational spectrum included small deletions, splice site mutations, and missense mutations, resulting in haploinsufficiency. Patients showed D4Z4 hypomethylation to levels less than 25% (normal being about 50%), and protein blot analysis in several patients showed decreased SMCHD1 protein in fibroblasts. Affected individuals were also heterozygous or homozygous for an FSHD1 (158900)-permissive D4Z4 haplotype that contains a polyadenylation signal to stabilize DUX4 mRNA in skeletal muscle. Primary myotubes from a normal individual with a normal-sized and methylated D4Z4 array on a permissive haplotype showed no DUX4 mRNA. However, decreasing SMCHD1 expression to about 50% using RNA interference resulted in transcriptional activation of DUX4 and a variegated pattern of DUX4 protein expression in the myotubes. The pattern of variegated DUX4 expression that resulted was similar to that observed in FSHD1 and FSHD2 myotube cultures. The findings indicated that SMCHD1 activity is necessary for D4Z4 hypermethylation and somatic repression of DUX4, and that reduction of SMCHD1 results in D4Z4 arrays that express DUX4 when a permissive haplotype is present. The SMCHD1 mutation and the permissive D4Z4 haplotype segregated independently in the families, indicating digenic inheritance. Of the 26 individuals with hypomethylation at D4Z4, a SMCHD1 mutation, and a permissive D4Z4 haplotype, 5 (19%) were asymptomatic, indicating incomplete penetrance.
In 2 cases of Ewing-like sarcomas (see 612219), Kawamura-Saito et al. (2006) identified the chromosomal translocation t(4;19)(q35;q13). The breakpoint at chromosome 19q13 was within exon 20 of the CIC gene (612082), and the breakpoint at chromosome 4q35 was within the DUX4 coding region in the D4Z4 repeat region. The translocation resulted in a CIC-DUX4 fusion transcript that was translated into a chimeric protein containing most of the CIC sequence, including the HMG box and TLE (see 600189)-binding sites, fused in frame to the C terminus of DUX4. The chimeric protein did not contain the N-terminal DNA-binding homeodomains of DUX4. No reciprocal DUX4-CIC transcripts were observed. The CIC-DUX4 transcript induced anchorage-independent growth when transfected into mouse fibroblasts. Although CIC is a transcriptional repressor, the CIC-DUX4 transcript enhanced transcription of a reporter gene when transfected into HeLa cells. Microarray analysis revealed altered gene expression following transfection of CIC-DUX4 into a human osteosarcoma cell line, including significantly upregulated expression of ERM (ETV5; 601600) and ETV1 (600541). Chromatin immunoprecipitation analysis and electrophoretic mobility shift assays confirmed binding of the chimeric protein to the ERM and ETV1 promoters.
Clapp et al. (2007) identified D4Z4 homologs in the genomes of rodents, Afrotheria (superorder of elephants and related species), and other species and showed that the DUX4 ORF is conserved. Phylogenetic analysis suggested that primate and Afrotherian D4Z4 arrays are orthologous and originated from a retrotransposed copy of an intron-containing DUX gene, DUXC. Reverse-transcriptase PCR and RNA fluorescence and tissue in situ hybridization data indicated transcription of the mouse array. Clapp et al. (2007) concluded that, together with the conservation of the DUX4 ORF for more than 100 million years, this strongly supports a coding function for D4Z4 and necessitates reexamination of the current models of mechanism for FSHD.
Dux, or Duxf3, is the mouse ortholog of human DUX4. Chen and Zhang (2019) found that both Dux zygotic knockout (Z-KO) and Dux maternal and zygotic knockout (MZ-KO) embryos were born at reduced mendelian frequencies but survived to adulthood without obvious abnormalities. RNA-sequencing analyses of 1-cell and late 2-cell Dux MZ-KO embryos revealed that loss of Dux had a minimal effect on zygotic genome activation (ZGA). Although Dux is essential for embryonic stem (ES) cells to enter the 2-cell (2C)-like ES cell state, most Dux targets in 2C-like cells were activated normally in MZ-KO embryos.
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